Thin-layer chromatography and determination of thiamine salts, phosphoric esters, disulphides, and their respective thiochromes

771

NOTES

Thin-layer

chromatography

esters, disulphides,

and detxwmination

and their

of thiamine

salts, phosphoric

respective thiochromes

A method for the routine determination

of thiamine salts, esters, disulphides as well as of their degradation derivatives for control of purity and stability of pharmaceutical preparations is proposed. Many separative techniques have been described previously, viz. paper electropkoresisl and chromatography’-8, ion-exchanger9 a-l2 and thin-layer chtomatographyl+le, but generally they are time-consuming or reliable only for qualitative purposes. We have developed a new method where thin-layer chromatography is associated with a calorimetric procedure, resulting in a fast qualitative and quantitative determination. Materials

San@les. The following were standardized commercial products : (s) Salts : thiamine hydrochloride and mononitrate. (2) Phosphoric esters : thiamine monophosphate as chloride and chloride-hydrochloride, phosphate and diphosphate salts; thiamine diphosphate (pyrophosphate) chloride (cocarboxylase) . (3) Disulphides : thiamine propyldisulphide hydrochloride and thiamine disulphide hydrochloride. Thiochrome derivatives of all the above compounds were prepared by oxidation in alkaline medium”, but with previous reduction in the case of thiamine disulphide and propyldisulphide. Thiamine triphosphate chloride was prepared according to BIGLINO AND SEGRIP and chlorothiamine according to SCWULTZ~~. CJtronaaiogra~Jaic tecJ&que Thin-layer plates were prepared with Silica Gel GF,,,, Merck (30 o/0 in ethanol 95”) and dried first in air and then in an oven at 40”. 50-100 ,ug of each sample in aqueous solution were spotted byamicrometric volume measuring device, and dried without heating.’ The solvent mixture diethanolamine-methanol-formic acid gg ~o-M/~g dibasic sodium phosphate (I : 15 : L .5 : 5) was used. The spots of the compounds were detected by a Mineralight SL 2537. The silica corresponding to each spot was quantitatively removed, weighed and used in the calorimetric assayo. Calorimetric

teclznique

Reagegzts. (I) Alkaline potassium ferricyanide solution : I vol. of I y. potassium ferricyanide added to g vol. of 20 yO sodium hydroxide is prepared just before use and kept in the dark. (2) Alcoholic hydrogen peroxide solution: 0~5 ml of 3 y. H,O, is added to go ml of ethanol 93” and brought to IOO ml with water. The suspension of the silica powder in 2.5 ml of O,I N HCl in a IO ml volumetric flask is thoroughly shaken at frequent intervals.over ~5 min. 2.5 ml of reagent, (I) are added, the mixture is shaken and, after one minute, it is made up to volume ‘with reagent (2). The flask is closed and shaken until decoloration is achieved; then it is opened to allow the gas to escape, and the volume is re-adjusted with reagent (i). J, Cltrovnatog., 32 (1968) 771-773